Mass spectrometry is used to measure the mass of a sample molecule, as well as the mass of the fragments of a sample molecule to identify that sample. The simplest mass spectrometers introduce a gaseous, electrically neutral sample into a vacuum, normally at pressures of 10−6 torr or less. Silverstein, et al, Spectrometric Identification of Organic Compounds, p. 7 (John Wiley & Sons, Inc. 1963). The sample then passes through an electron beam.
The fast-moving electrons from the electron beam strike electrons on the sample being studied, ejecting one or more electrons from the sample. After a subject sample molecule has lost an electron, the sample has a net positive charge, or is “ionized.”
Mass spectrometry measures the ratio of the mass of the molecule to the ion's electric charge. The mass is customarily expressed in terms of atomic mass units, called Daltons. The charge or ionization is customarily expressed in terms of multiples of elementary charge. The ratio of the two is expressed as a m/z ratio value (mass/charge or mass/ionization ratio). Because the ion usually has a single charge, the m/z ratio is usually the mass of the ion, or its molecular weight (abbreviated MW). Often, the terms m/z, the mass of the sample in Daltons (or molecular weight, abbreviated MW) are used interchangeably.
Molecules that are not easily rendered gaseous are more difficult to study with mass spectrometry. Accordingly, many modern advances in mass spectroscopy address problems regarding the handling of liquid or solid samples. When a molecule is ‘on’ a substrate, the sample is adsorbed to that substrate. Desorption is the process by which a molecule adsorbed on a substrate is removed from the substrate. Removing a molecule from a surface is “desorbing” a molecule from that surface. When desorbed, the molecule may be “vaporized”, that is rendered into a gaseous state. Instead of starting with a gaseous sample, as basic mass spectrometry does, desorption mass spectrometry starts with the sample adsorbed on a substrate and desorbs the sample, thereby providing the gaseous sample to the mass spectrometer.
A desorption mass spectrometric methodology used for analysis of biological samples is matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). In conventional MALDI, the sample is typically dissolved into a solid, ultraviolet-absorbing, crystalline organic acid matrix. The liquid matrix/sample is deposited onto a inert base plate in a specific pattern and allowed to dry into target spots. Pulsed laser radiation ionizes some sample molecules while vaporizing the spot carrying the sample with the vaporized matrix. Many MALDI instruments are preconfigured to ionize the spots in sequence to maximize throughput of the system.
MALDI-MS allows for desorption and ionization of non-volatile samples (e.g., biological samples) from a solid-state phase directly into the gas phase without charring, fragmentation, or chemical degradation. MALDI-MS is used to analyze substances such as polypeptides, polynucleotides, proteins, DNA fragments, biopolymers, and other large molecules. The development of proton transfer ionization has made biomolecular mass spectroscopy possible.
MALDI is severely limited in the study of small molecules. The MALDI matrix interferes with measurements below a m/z of approximately 700, called the low-mass region, which varies somewhat depending on the matrix used. Even with large molecules, MALDI has significant limitations. The matrix and matrix fragments can form adducts with the sample ion that can cause the measured signal to have a range of molecular weights. A spectrum from such an analysis may have substantially shortened peak heights.
New methods of desorbing samples have been developed that utilize a substrate to hold the sample rather than having the sample be mixed with a matrix and then be adsorbed to the base plate as was previous practice with MALDI spectrometers. Substrates of porous silicon have been used with laser equipped mass spectrometers to perform analyses of samples. As used herein, the term “DIOS” refers to desorption ionization on silicon, a structure that is described in U.S. Pat. No. 5,882,496 the contents of which is hereby expressly incorporated herein by reference in its entirety. A DIOS substrate (referred to as a chip) typically has dimensions of approximately three to five centimeters and a thickness of 0.5 millimeters. The sample, generally in the form of an aqueous solution in which one or more compounds are dissolved, is received on the porous silicon surface of the substrate. When used in the determination of mass and charge information of ions formed by laser ionization, the substrate is placed in close proximity to the inlet of a mass spectrometer. The laser is discharged or pulsed, ionizing and vaporizing a portion of the sample but leaving the substrate behind. The vapor, ions and gases are drawn into the inlet of the mass spectrometers for analysis.
The DIOS methodology provides the beneficial effects of having a direct laser desorption/ionization technique for use in biomolecular and other analyses. The DIOS methodology addresses the unfulfilled needs of the present methods for dramatically simplified sample preparation. Sample preparation is simplified because of the absence of the matrix or the need for covalent linkage of the analyte to the substrate. Further, substrates do not need to be tailored to the needs of a particular sample, and the DIOS method has a tolerance for salts and buffers.
In order to utilize the DIOS chip with existing MALDI instruments, ways need to be developed to secure the substrates in the instruments without introducing chemicals. It would be preferable to have the chips positioned repeatably so that the instrument does not need to be retargetted between chips. Further, when the analysis of the samples on the substrate is complete, an easy way to remove and dispose of used substrate would be useful. The present invention addresses these needs.